The role of Lck kinase in T cell activation is well established. The key step in the TCR proximal activation signals mediated by Lck is the phosphorylation of immunoreceptor tyrosine based activation motifs (ITAM) present in the cytoplasmic tails of CD3 chains and the phosphorylation of ZAP-70 kinase. The inhibition of Lck action hampers these initial events preventing the activation of multiple signaling pathways required for IL-2 production and antigen-specific T cell proliferation, thus blocking immune response. Due to its pivotal role in T cell activation and due to its restricted expression, Lck is an attractive molecular target for the development of novel immune response inhibitors.
Lck is a Src family kinase composed of several distinct functional domains, the kinase, SH2 and SH3 domains. Among these functional domains, the kinase and the SH2 domains are attractive targets because of their roles in catalytic action and in defining specificity. Chemical targeting of the Lck kinase domain has been used to achieve a therapeutic outcome. Lck kinase catalytic inhibitors so far developed lack desired specificities and exhibited severe toxicity in vivo making them unsuitable for further drug development. Since similar Src family kinases such as Fyn and Lyn are expressed by lymphoid cells, development of inhibitor compounds that specifically target the Lck kinase is essential. All of the kinase catalytic inhibitors are ATP analogues and lack the desired level of specificity towards other closely related Src family kinases, especially Lyn.
Since SH2 is a functional domain that binds to specific tyrosine phosphorylated sites of proteins, studies have been focused on the relevant portions of the SH2 domain structures, i.e., the region to which the phosphorylated tyrosine (pY) target peptides bind. 3D structural analyses have shown that the binding domain is comprised of a crevice along the surface of the protein to which the phosphopeptide binds in an extended conformation. The crevice includes two well-defined binding pockets for the pY residue and for the pY+3 residue. The binding specificity of SH2 domains are primarily dictated by binding sites that interact with the amino acid residues on the C-terminal side of the pY, especially the third residue (pY+3 sites). Lck has been shown to bind with the highest affinity to the ζ chain ITAM-2 C terminal phosphotyrosine residues. Such selectivity of SH2 domain is essential for a proper cellular activation and suggests that SH2 domains may be potential targets for the design of compounds that specifically inhibit selected signal transduction pathways. Blocking the association of Lck SH2 domain with the CD3 ITAM prevents T cell activation. Accordingly, Lck SH2 domain is a potential target for immunosuppressive drug development.
Based on the vast knowledge of SH2 domain 3D structure and the sequence specificity of SH2 domains, rational design of peptides and peptidomimetics that are specific for individual SH2 domains has been performed. However, these peptidomimetics presented problems of stability and permeability that limited their further development into therapeutic agents. To address this limitation, the discovery of small molecular-weight non-peptidic compounds targeted towards the pY+3 binding pocket that specifically block SH2 domain binding to its cellular target protein has been carried out as an approach for the development of novel immunosuppressants.
SH2 domains have been the target of a large number of 3D structural studies as well as computational studies via molecular simulations. Currently, there are over 220 structures available in the NCBI Protein Data Bank that include SH2 domains alone and in complex with various peptides. Using the available structural information of Lck SH2 domains in the database, computer aided drug design (CADD) was used to identify small molecule inhibitors of Lck SH2 (SMILS) targeting the pY+3 binding pocket. Three compounds were identified to be selective in inhibiting Lck SH2 domain binding to the CD3 ζ chain ITAM. These SMILS inhibited IL-2 production, mixed lymphocyte reaction in vitro and popliteal lymph node assay in vivo.
Rheumatoid arthritis (RA) is a chronic multi-system T cell autoimmune disease. Several lines of evidence derived from research on the rat adjuvant arthritis (AA) experimental model support an etiologic relationship between AA and chronic inflammatory RA in humans. For example: a) in RA patients, an association between T cell responses to mycobacterial hsp65 (Bhsp65) and early stages of joint inflammation has been noted; b) T cell responses to Bhsp65 are elevated in RA synovial fluid compared with blood; c) RA patients raise T cell responses not only to native Bhsp65 but also to its peptide 180-188 (the arthritogenic determinant for AA in the Lewis rat); d) arthritis patients with juvenile chronic arthritis also raise vigorous T cell responses to BhspB5 and self hsp65. Thus, T cell responses to Hsp antigens are involved in the disease process in RA. Among the subsets of T cells, it is believed that Th1 cells are involved in the pathogenesis of T cell autoimmune diseases. Shifting of differentiation of autoreactive T cells towards the Th2 effector phenotype is associated with the improvement of RA.
The predominantly T cell-mediated nature of the disease process, the well-established experimental model of AA in Lewis rat that closely resembles human RA, the well-defined pathogenic epitope within Bhsp65, the well-characterized pathogenic epitope-specific T helper 1 (Th1) subset, and the AA-protective attribute of anti-Bhsp65 antibodies provide unique opportunities for the validation and the analysis of the mechanism of action of SMILS.
Upon antigen-mediated activation, T cell antigen receptor complex becomes phosphorylated at ITAM residues. Although there are numerous signaling proteins with SH2 domains in T cells, only a few bind to the ITAM via their SH2 domains. Thus, in phosphotyrosine based signaling events, the specificity is determined by the SH2 domain. A successful strategy to identify small molecule inhibitory compounds targeting the SH2 domain pY+3 binding pocket using combination of CADD and in vitro solid phase EIA screening methods has been developed. Among the compounds selected in the screening process, compounds have been identified that have restricted specificity, only inhibiting the association of Lck SH2 domain with the CD3 ζ chain ITAM but not the relatives of Lck in the Src family such as Fyn and Lyn. Identification of small molecule inhibitors that can discriminate between such closely related SH2 domains is novel. The striking specificity of these compounds towards Lck SH2 domain, their permeability and low toxicity make them attractive as immunosuppressants.
The present invention provides specific non peptide small molecule inhibitors that block Lck SH2 association with the tyrosine phosphorylated ITAMs of CD3 chains. These compounds (small molecule inhibitors of Lck SH2 domain:SMILS) specifically inhibit Lck function and block IL-2 production by activated T cells, mixed lymphocyte reaction in vitro as well as local allogeneic response of popliteal lymph node assay in vivo and mycobacteria-induced (Mtb-induced) adjuvant arthritis in rats. Compounds are cell permeable, non-cytotoxic and well tolerated by animals without any visible toxic effects. These compounds specifically suppress T cell activation and reduced joint inflammation in arthritic rats. Immunosuppressors can be used in the treatment of severe rheumatoid arthritis.